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TheJournalofClinicalInvestigation http://www.jci.org Volume 119 Number 9 September 2009 2455

A decade of molecular pathogenomic analysis of group A Streptococcus

James M. Musser1 and Samuel A. Shelburne III2

1Center for Molecular and Translational Human Infectious Diseases Research, The Methodist Hospital Research Institute, and Department of Pathology, The Methodist Hospital, Houston, Texas, USA. 2Department of Infectious Diseases, Division of Internal Medicine,

MD Anderson Cancer Center, Houston, Texas, USA.

MolecularpathogenomicanalysisofthehumanbacterialpathogengroupAStreptococcushasbeenconductedforadecade.Muchhasbeenlearnedasaconsequenceoftheconfluenceoflow-costDNAsequencing,microarraytech-nology,high-throughputproteomics,andenhancedbioinformatics.Thesetechnicaladvances,coupledwiththeavailabilityofuniquebacterialstraincollections,havefacilitatedasystemsbiologyinvestigativestrategydesignedtoenhanceandaccelerateourunderstandingofdiseaseprocesses.Here,weprovideexamplesoftheprogressmadebyexploitinganintegratedgenome-wideresearchplatformtogainnewinsightintomolecularpathogenesis.Thestudieshaveprovidedmanynewavenuesforbasicandtranslationalresearch.

IntroductionGroup A Streptococcus (GAS) is a Gram-positive bacterium that causes several diseases in humans, including pharyngitis and/or tonsillitis, skin infections (including impetigo, erysipelas, and other forms of pyoderma), acute rheumatic fever (ARF), scar-let fever, poststreptococcal glomerulonephritis (PSGN), a toxic shock–like syndrome, and necrotizing fasciitis (NF) (1–3). The organism is a human-adapted pathogen, that is, humans are the natural host and there is no animal or environmental reservoir that contributes to the life cycle of GAS. On a global basis, ARF and the rheumatic heart disease that follows are the most com-mon cause of preventable pediatric heart disease worldwide (4). There are 25–35 million cases of GAS pharyngitis per year in the United States, which are responsible for about 1–2 billion dollars per year in direct health care costs, and 600 million cases globally each year (Table 1) (2, 3). The continued high morbidity and mor-tality caused by GAS in developing nations, the substantial finan-cial burden attributable to GAS-related health care costs in the United States, the development and spread of antibiotic resistance in this pathogen (5–7), and the lack of a licensed human vaccine (8, 9) highlight the need for a fuller understanding of the molecu-lar pathogenesis of GAS. Especially vital is the need for informa-tion bearing on the molecular events contributing to pathogen-host interaction, clone emergence, and epidemics.

In recent years, a paradigm shift has occurred in the manner in which microbial pathogenesis problems are studied. The conflu-ence of data derived from genome-sequencing projects and the development of microarray technology permit a genome-wide strategy to be used for pathogenesis research, now commonly referred to as molecular pathogenomics investigation (10). Com-parative genome sequencing and other types of analyses of mul-tiple isolates of the same pathogenic bacterial species have revealed unexpectedly large amounts of intraspecies variation in gene content and uncovered strategies used in genome diversification (11–19). Strains of some bacterial species differ in gene content by up to 25% and have extensive allelic variation (11, 13, 15). This striking magnitude of genetic differentiation provides tremendous adaptive flexibility and influences the spectrum of clinical disease

caused by distinct clones of the species. For example, the genome of E. coli serotype O157:H7, which is responsible for hemolytic uremic syndrome, is approximately 25% larger (i.e., approximately 1 Mb of DNA larger) than that of many other strains of E. coli that do not cause significant disease in humans (13).

A molecular pathogenomics approach has been applied to GAS for nearly a decade and has yielded new information about the genetic basis of GAS pathogenesis, clone emergence, and strain genotype–disease phenotype relationships. Herein we highlight key findings that illustrate that molecular pathogenomics can be used to greatly accelerate the rate of obtaining new information about long-standing and previously intractable infectious disease ques-tions. Only by applying hypothesis-driven research and using new technologies will the elucidation of molecular events underlying infectious disease processes proceed with maximum efficiency.

GAS genome sequencesGeneral concepts. The full-genome sequences that are now available for 13 strains of GAS make it one of the more deeply sequenced species of human pathogens (15, 20–28). GAS strains can be clas-sified by either M protein or T protein serotype, depending on the composition of bacterial antigens expressed on their cell surface, and the 13 genome sequences represent serotypes responsible for more than 70% of M protein serotypes that commonly cause GAS pharyngitis and invasive infections in the Western Hemisphere (29–31). The strains selected for sequencing were chosen mainly because they represent abundantly occurring M protein sero-types or because they had a distinct clinical phenotype of special interest, such as high virulence, antimicrobial agent resistance, or association with a particular disease (15, 20–28). Several common themes have emerged from comparison of the genome sequences (15). Each genome is approximately 1.9 Mb in size, with approxi-mately 10% of the overall gene content encoded on variably present exogenous genetic elements such as prophages and integrated con-jugative elements (15), with the former accounting for most of the variably present gene content (15, 20–28). Importantly, the marked heterogeneity of gene content is even observed among GAS strains of the same M protein serotype. This extensive variation in gene content stands in stark contrast to humans and many other higher eukaryotic species, which have less than 0.1% variation in gene con-tent among different members of a species. The 13 GAS genome

Conflictofinterest: J.M. Musser received research support from Novartis Vaccines.

Citationforthisarticle: J. Clin. Invest. 119:2455–2463 (2009). doi:10.1172/JCI38095.

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2456 TheJournalofClinicalInvestigation http://www.jci.org Volume 119 Number 9 September 2009

sequences provide a critical resource for initiating investigation into how variation in gene content influences pathogenesis.

Discovery of new virulence factors through whole-genome sequence analy-sis. As recently as a decade ago, only relatively few GAS virulence factors had been identified. These included M protein, hyaluronic acid capsule, and the extracellular cysteine protease streptococcal pyrogenic exotoxin B (SpeB). Within a few years of the first GAS genome sequence becoming available, no fewer than 13 new pro-teins that contribute to pathogenesis had been described (32–45). Novel pathways of GAS host-pathogen interaction also had been elucidated. For example, Edwards et al. demonstrated that super-natants derived from GAS cleaved IL-8, resulting in reduced neu-trophil activation and migration (37). Using the GAS genome data, the IL-8–degrading activity was shown to be encoded by a previously uninvestigated gene (spy0416, using the serotype M1 numbering system for strain SF370), and the proteinase encoded by this gene was subsequently termed “Streptococcus pyogenes cell envelope proteinase” (SpyCEP) (46, 47). In a short time, it has been learned that SpyCEP-mediated cleavage of IL-8 decreases neutro-phil endothelial transmigration and that degradation of addition-al chemokines by SpyCEP retards neutrophil activation (46–48). Together, these discoveries indicate a key role for SpyCEP in GAS host-pathogen interaction (46–50). Similar rapid progress, facili-tated by the availability of the GAS genome sequences, has been made in identifying and characterizing the function of novel GAS immunoglobulin-degrading enzymes (39, 40, 51), collagen-like proteins (41, 44, 52), and several superantigens (53–55). Given that numerous open reading frames in the GAS genome that encode putative cell surface proteins have yet to be investigated, it seems highly likely to us that heretofore unidentified GAS virulence fac-tors will continue to be discovered in the coming years.

Expanded understanding of virulence factor regulation pathwaysAs noted above, the GAS genome encodes a broad range of viru-lence factors that are critical to the diverse array of infections that the bacterium causes (1). One important impact of GAS genome-wide investigations has been to provide an enhanced molecular

understanding of how the pathogen coor-dinates virulence factor production (56). There are 13 conserved two-component regulatory systems (TCSs) in the complet-ed GAS genomes, only one of which had been studied prior to the availability of the first GAS genome sequence in 2001 (57). TCSs regulate multiple unlinked chro-mosomal genes and control coordinated expression of genes encoding virulence fac-tors, such as toxins, degradative enzymes, and immune-modulating molecules, in response to environmental stimuli. Since 2001, each of the 13 TCSs has been studied by genome-wide analyses to some degree, many in detail (58–62). Similarly, there are more than 100 putative stand-alone transcriptional regulators encoded within the GAS genome, only a few of which had been identified before the availability of a genome sequence. Of the 100 putative stand-alone transcriptional regulators, 12

have now been investigated in detail, and the transcriptomes that eight of these regulate in vitro have been determined (17, 63–68).

Genome-wide investigations of GAS regulatory pathways have transformed our understanding of pathogenesis. A key finding has been that global gene transcription varies highly depending on the environment, growth conditions, and stage of growth of the bacterium (69–72). For example, Voyich et al. (73) have shown that the GAS transcriptome is substantially altered in response to phagocytosis by human polymorphonuclear leukocytes (PMNs), a key step in invasive GAS disease. Similarly, interaction with pharyngeal epithelial cells and human saliva was found to induce marked alterations in the GAS transcriptome, thereby providing new information about potential strategies used for infection of and persistence in the oropharynx (69, 74). Longitudinal analysis of changes in the GAS transcriptome over time in nonhuman primates has revealed that the temporal pattern of GAS gene transcription in pharyngitis is very closely linked to three dis-tinct phases of infection, namely colonization, acute infection, and asymptomatic carriage (75). During the colonization phase, when GAS CFUs were low, the expression of genes involved in carbohydrate metabolism was greatly increased, suggesting that carbon source acquisition is a key step in initial GAS growth and establishment of infection. Expression of genes encoding GAS virulence factors with known roles in GAS survival, dissemina-tion, inhibition of PMN recruitment, and induction of host cytokines were highly expressed in the acute phase of infection, concomitant with an increase in GAS CFUs and host inflamma-tion. Thus, the in vivo infection data are in strong alignment with ex vivo results (i.e., those obtained using saliva, epithelial cells, and PMNs).

A key finding of repeated GAS transcriptome analyses has been the elucidation of previously unappreciated connections between distinct gene categories. For example, recent work has shown that transcript levels of genes encoding proteins involved in carbohy-drate catabolism and genes encoding virulence factors change in concert in response to environmental stimuli (74, 75). Such changes are at least partially due to a regulatory circuit controlled by catabolite control protein A (CcpA) (Figure 1) (59, 74–78). Simi-

Table 1Minimum summary estimates of the global burden of GAS diseases

Existingcases Annualnewcases Annualdeaths

SevereGASdiseaseRHD 15.6 million 282,000 233,000History of ARF without carditis, 1.88 million 188,000 — requiring secondary prophylaxisRHD-related infective endocarditis — 34,000 8,000RHD-related stroke 642,000 144,000 108,000Acute poststreptococcal — 472,000 5,000 glomerulonephritisInvasive GAS diseases — 663,000 163,000Total severe cases 18.1 million 1.78 million 517,000

SuperficialGASdiseasePyoderma 111 million — —Pharyngitis — 616 million —

Table 1 is reproduced with permission from The Lancet Infectious Diseases (3). RHD, rheumatic heart disease. —, data not provided in the original reference.

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larly, a genome-wide investigation of SpeB regulation led to the discovery that a lactose catabolism enzyme (LacD.1) has evolved to coordinate alterations in GAS virulence factor production as a result of changes in carbon source availability (Figure 1) (79). Anal-ysis of completed GAS genomes led to the discovery that one of the two GAS lactose operons has retained a catabolic role, whereas the other has evolved a regulatory function (Figure 1) (80).

In addition to discovering ties between central metabolic pro-cesses and pathogenesis, GAS genome-wide studies have also revealed interactions between metal regulation, oxidative stress, and pathogenesis. Analysis of the completed genomes indicates that GAS encodes two highly conserved metalloregulators, MtsR and PerR, that regulate proteins involved in iron and manganese uptake (81, 82). Animal studies have found that PerR is needed for full GAS virulence in skin and soft tissue infection and in oro-pharynx infection (65, 82, 83). Surprisingly, the PerR regulon was found to include numerous carbohydrate utilization genes, sug-gesting links among central metabolic processes, oxidative stress response, and virulence (65). The key role for MtsR in the develop-

ment of NF is discussed below in “Genome-wide dissection of the molecular events underlying GAS epidemics.”

Insights into the molecular basis of GAS disease specificityInvestigations into the molecular basis of GAS host site predilection. The two major sites of GAS infection are the human throat and skin (84). It has long been recognized that particular M protein sero-types mainly cause pharyngitis, whereas others predominate in skin infection, leading to the idea of skin-specialist and throat-specialist GAS strains (85, 86). However, the molecular basis for these observations has been unclear (86). Bioinformatic study of GAS genomes led to the identification of an area of the genome that is highly variable between M protein serotypes, referred to as the fibronectin-binding, collagen-binding T antigen (FCT) region (85). Lately, evidence has accumulated that genetic heterogeneity within the FCT region may be a major factor determining why par-ticular GAS strains colonize and infect distinct host regions (87). For example, the FCT region contains genes encoding the recently

Figure 1Schematic depicting how genome-wide analyses have revealed previously unsuspected links between carbohydrate metabolism and virulence. Examples of recently discovered relationships between carbohydrate catabolism and virulence include (i) the role of LacD.1 in controlling SpeB production, (ii) identification of the SptR/S saliva persistence pathway, (iii) the discovery of regulation of virulence factors by CcpA, and (iv) the dif-ferential role in virulence factor regulation (bottom) and catabolism (top) of the 2 lactose operons. Panel i is reprinted with permission from EMBO Journal (79). Panels ii and iii are adapted from Proceedings of the National Academy of Sciences U. S. A. (ref. 75 and ref. 77, respectively).

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discovered cell surface pili that are critical to GAS epithelial cell adhesion (32, 33, 36). Thus, strain-to-strain differences in pili com-position may contribute to the predisposition of particular GAS M protein serotypes for certain host sites in an analogous fashion to that observed for E. coli (88). Elucidation of the molecular basis for why particular GAS strains colonize and infect particular host environments holds the promise for developing novel preventive and therapeutic targets.

New insights into relationships between mucosal and invasive GAS dis-ease. GAS thrives at human mucosal sites and also causes devas-tating invasive infections. Strains isolated from mucosal sites are genetically indistinguishable from invasive strains by standard assays, such as M protein serotyping and multi-locus sequence typing (89, 90). However, these techniques index only a very small part of the genome, which means that they greatly underestimate the amount of genetic variation present. Using newly developed genome resequencing techniques, Sumby et al. (91) analyzed the

complete genomes of GAS isolates recovered from the spleen of mice that had been infected subcutaneously. Compared with the strain used to inoculate the mouse skin, the invasive GAS isolates (i.e., those obtained from the spleen after skin inoculation) had mutated forms of the control of virulence (CovR/S) TCS (91). Mutation of this TCS resulted in derepression of numerous viru-lence factors critical for combating the host immune system com-ponents encountered during bloodstream infection (Figure 2) (91). For example, GAS secretes a potent DNase that is involved in escape from neutrophil extracellular traps (NETs), a host immune defense mechanism generated by dying PMNs (92). The DNase is upregulated in CovR/S mutants, thereby contributing to the devel-opment of invasive disease. The clinical relevance of these findings was confirmed by the discovery that many GAS strains causing invasive infections in humans often have function-altering muta-tions in the genes that encode the components of the CovR/S TCS (91, 93). Thus, it is currently thought that GAS mucosal isolates have an intact CovR/S system that limits GAS virulence factor pro-duction, whereas interaction with the host immune system and/or deep tissues selects for strains with CovR/S mutations, leading to a hypervirulent phenotype and the serious manifestations of inva-sive GAS disease (Figure 2).

Establishment of the key role of lateral gene transfer in the emergence of unusually virulent clonesSeveral types of molecular events contribute to the evolution and emergence of bacterial strains with enhanced virulence. The most well-understood and by far the most studied process is horizon-tal gene transfer (HGT), which involves bacterial viruses known as bacteriophages (transduction), plasmids (conjugation), and genomic DNA (transformation) (16, 94). HGT events create new strain genotypes by moving blocs of genetic material — sometimes large pieces of DNA that exceed 40–60 kb in size — between strains. Thus, HGT events represent a quantum evolutionary leap that can increase bacterial fitness by enhancing antimicrobial agent resistance, immune avoidance, and capacity to colonize or infect a new ecological niche. As in eukaryotes, bacterial evolution also occurs by more subtle processes, including accumulation of point mutations and small genomic changes such as those created by slipped-strand mispairing. Several of these molecular processes have contributed to the recent emergence and intercontinental dissemination of a new clone of serotype M1 GAS with distinct virulence properties (22, 95–97). This understanding was revealed by several lines of work, including comparative genome charac-terization conducted in several laboratories in a span of almost

Figure 2The role of the CovR/S TCS in the transition from mucosal to invasive disease. (A) Upon entering subcutaneous tissue, GAS is confronted by neutrophils that produce NETs. (B–D) Mutation in the CovR/S TCS (B) allows for high-level production of a GAS DNase that degrades NETs (C), thereby increasing pathogen survival and enhancing the probability of invasive infection (D). (E) Principal component analysis of expression microarray data demonstrates that mucosal (or pha-ryngeal) and invasive GAS isolates have strikingly different transcrip-tomes. (F) Examples of genes encoding critical virulence factors that are more highly expressed in invasive isolates (red) or pharyngeal isolates (blue). ITP, invasive transcriptome profile; PTP, pharyngeal transcriptome profile. A–D are reprinted with permission from Nature Medicine (93). E and F are adapted from PLoS Pathogens (92).

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20 years (22, 46, 95, 96, 98, 99). Comparative pathogenomic analy-sis resulted in two particularly important findings (22). First, low- and high-virulence serotype M1 strains differ in bacteriophage content and chromosomal integration site (22). Second, it was unexpectedly also discovered that another HGT event, involving reciprocal recombination of a 36-kb chromosomal region encod-ing the secreted toxins streptolysin O and NAD+-glycohydrolase, was a critical evolutionary event that shaped the genome of con-temporary virulent M1 strains (22). The likely mechanism under-lying this event was generalized transduction, a process involving inadvertent packaging of a random chromosomal segment from a donor strain into a bacteriophage capsid head, followed by transfer to a new recipient bacterial strain. Importantly, contemporary vir-ulent M1 strains produce high levels of these two toxins compared with older, less virulent M1 strains, but the underlying molecular mechanism of increased expression is not yet understood.

Host susceptibility to invasive GAS diseaseLike other infectious diseases, the clinical manifestations of GAS infection reflect interaction between the bacterium and the host. In contrast to situations in which infection with the pathogen is the prime determinant of disease, such as occurs for Bacillus anthra-cis or ebola virus, most humans are repeatedly exposed to and colo-nized by GAS without developing clinical symptoms. Moreover, GAS isolates that cause superficial infections such as pharyngitis and impetigo can be genetically closely related to those of the same M protein serotype that cause lethal infections such as NF and

toxic shock syndrome (90, 100). Therefore, it is highly probable that the development of severe clinical manifestations following GAS infection has a strong host susceptibility component. Most investigations of host susceptibility to severe GAS infection have focused on the role of HLA polymorphisms (101, 102). The inter-action of GAS superantigens with HLA class II molecules on anti-gen-presenting cells can result in the activation of up to 25% of all T cells at a given time, although the number of T cells activated by a particular GAS superantigen varies substantially from per-son to person (103, 104). The protective and deleterious roles of particular HLA alleles in the development of streptococcal toxic shock syndrome were observed in an epidemiologic investigation and confirmed using transgenic mice (101, 102, 105).

In addition to focused research on HLA polymorphisms, recent studies have also begun to dissect the molecular basis of host susceptibility to serious GAS infection in mice using a genome-wide approach (106, 107). Researchers have exploited differences in susceptibility to GAS infection among strains of genetically defined mice to begin to localize protective and deleterious host genetic polymorphisms (106, 107). For example, a heightened inflammatory response to GAS was associated with worse out-comes in a mouse model of infection and linked to alterations in the expression of genes involved in apoptosis, macrophage activation, and prostaglandin synthesis (106). A genome-wide transcriptome analysis of murine macrophages also identified genes encoding proteins involved in prostaglandin synthesis as being upregulated during interaction with GAS, and the use of

Figure 3Molecular pathogenomic analysis reveals the contribution of a HGT event to the evolution of a female urogenital specialist GAS clone of serotype M28. The 37-kb region of difference 2 (RD2) element is shared among GBS strains and serotype M28 GAS. An HGT event may have occurred directly between a GAS and GBS strain, or an unknown third-party donor may have participated. Adapted with permission from The Journal of Infectious Diseases (23) and Molecular Microbiology (125).

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inhibitors of prostaglandin synthesis has been associated with severe GAS infection in humans (108, 109). There are significant experimental design and execution barriers to extrapolating the genome-wide approach to enhance understanding of human sus-ceptibility to GAS infections. However, the key point is that the increasing availability of tools such as genome-wide SNP analysis holds significant promise for increasing our understanding of human host determinants of GAS infection.

Serotype M28 and puerperal sepsis: pathogenesis consequences of a chimeric genomeAlthough group B Streptococcus (GBS) is a well-known cause of seri-ous neonatal or maternal infections, GAS can also be responsible for these infections. Genome sequencing and pathogenesis stud-ies have provided unexpected clues as to why serotype M28 GAS strains are repeatedly overrepresented in puerperal sepsis (child-bed fever) cases (23).

It was hypothesized (23) that analysis of the genome sequence of a serotype M28 GAS strain causing puerperal sepsis would iden-tify novel genetic elements that contributed to the overrepresenta-tion of strains of this serotype in this infection type. Analysis of the genome sequence of a serotype M28 strain has borne this out, providing the highly unexpected discovery that these GAS strains have a genome that is a chimera, composed of largely GAS genetic material onto which has been molecularly grafted a large piece of foreign DNA shared with GBS strains (23) (Figure 3). The foreign DNA is 37.4 kb in size, was acquired by HGT, and encodes seven secreted proteins that are produced in human infections (35). With very few exceptions, this genetic element is not present in other GAS strains. Importantly, one of the secreted proteins medi-ates attachment of M28 strains to human urogenital epithelium; another binds to GP340, a large human glycoprotein abundantly present in vaginal and oral secretions (35, 110). Thus, the M28 genome has been shaped by acquisition of a foreign genetic ele-ment that assisted in creating a disease-specialist GAS strain. As with other GAS molecular pathogenomics studies, these discover-ies have served to catalyze downstream pathogenesis experiments. Studies are ongoing that are designed to provide a deeper under-standing of the precise molecular processes involved in this niche adaptation and subsequent infection.

Genome-wide dissection of the molecular events underlying GAS epidemicsGAS has been used as a model system to study the molecular pro-cesses contributing to epidemics. Since 1992, more than 350 sero-type M3 strains have been recovered in a prospective population-based surveillance study of GAS invasive infections being conductedin Ontario, Canada (27, 111, 112). These strains have caused two temporally distinct epidemic waves, centered in 1995 and 2000 (19). A molecular pathogenomics approach allowed the identification of key contributors to the episodic behavior of the GAS invasive iso-lates. First, the distinct epidemics were shown to be caused by a het-erogeneous array of serotype M3 subclones, rather than recycling of a single clone (19). These distinct clones were characterized by the acquisition or loss of specific prophages that encode known GAS virulence factors. Second, host selective pressure appears to have resulted in a highly successful clone that rose to dominance in the second epidemic wave. DNA sequence analysis, coupled with immu-nologic studies, identified a four–amino acid duplication in the amino terminus of M protein in the new subclone responsible for

many of the invasive cases in the 2000 epidemic (19). This duplica-tion resulted in alterations in linear B cell epitopes, which produced substantial differences in the ability of human PMNs to phagocyto-size and kill strains with the variant M protein (19). This key finding indicated that subtle or relatively minor allelic variation may partici-pate in clone emergence and perpetuation of the epidemics.

To identify microbial genes or specific allelic variants that influ-ence the outcome of host-pathogen interactions, it is possible to use a strategy analogous to that commonly practiced when under-taking genome-wide association studies (GWASs) — using genetic methods such as high-density SNP analysis — on humans with particular disease phenotypes, such as type 2 diabetes mellitus, macular degeneration, and schizophrenia (113). In many regards, GWASs are considerably simpler in bacterial infectious disease studies because, compared with humans, prokaryotic organisms have very small genomes and isogenic mutant strains can be gener-ated and used to confirm the findings from the genetic association study. A novel bacterial GWAS conducted on strains recovered in the Ontario GAS epidemics (17) led to the discovery that a single nucleotide mutation in the gene encoding the MtsR metalloregu-latory protein implicated in uptake of iron or manganese results in a decrease in the ability of GAS to cause devastating NF. The mechanism underlying this decreased ability to cause severe dis-ease involves dysregulation of the control circuit responsible for wild-type levels of SpeB (our unpublished observations), a potent broad-spectrum protease that degrades extracellular matrix pro-teins, inactivates innate immune molecules, and destroys host tis-sue (114, 115). Inasmuch as single nucleotide mutations are the most abundant cause of genetic variation among members of the same species (13–15, 17, 18, 116), this discovery has broad implica-tions for the confluence of bacterial molecular population genom-ics and pathogenesis research.

Accelerated vaccine candidate identification and developmentIn the long term, the key to the control of most human pathogens is the use of efficacious vaccines, and GAS is no exception. Trans-formation of vaccine research and development by genome-wide analyses is expertly summarized in this Review series by Rinaudo et al. (117). Nevertheless, we believe that a few points related to GAS vaccine research need to be stressed here. Although the quest for a successful human vaccine against GAS pharyngitis and inva-sive infections has been ongoing for many decades (reviewed in ref. 8), and much progress has been made, a product licensed for use in the United States or elsewhere is still lacking. The many complete genome sequences of GAS that are publicly available have been used to assist the decades-long search for GAS vaccine candidates. For example, Lei et al. (118) analyzed the extracellular proteome and identified several previously undescribed proteins that have been the subject of subsequent vaccine research. Simi-larly, bioinformatic analysis of multiple GAS genomes identified 16 highly conserved cell surface lipoproteins. Subsequent bio-chemical, immunological, and mouse model research identified five lipoproteins as potential new vaccine candidates (119). Using a proteomics approach, Cole et al. showed that the GAS cell surface contains numerous immunogenic proteins not previously sus-pected of being associated with the cell wall, thus expanding the number of potential GAS vaccine candidates (120). The finding of proteins on the GAS cell surface or in the culture supernatant that lack traditional motifs associated with cell surface and secreted

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proteins has been a consistent finding of proteomic studies, stress-ing the need for experimental validation of predictions based on bioinformatic analyses (118, 120–123). Improved modalities for separating GAS cellular versus cell surface constituents may pro-vide additional insights into novel GAS proteins that are critical for host-pathogen interaction and are thus potential vaccine can-didates (124). In the most extensive GAS vaccine candidate study to date, Rodriguez-Ortega et al. (50) analyzed the surface-exposed proteome of GAS and identified one new antigen (SpyCEP) that conferred the ability to protect mice against lethal infection.

What does the future hold?Enormous strides have been made in the last decade in our under-standing of GAS-host interactions, and molecular pathogenomics studies have contributed substantially. However, many gaps remain in our knowledge. Of note, many aspects of the host response dur-ing GAS infection remain largely unknown, but application of the genome-wide integrative strategies discussed above to the host side of the equation are likely to yield useful data. Analysis of patients with ARF may be particularly useful in this regard, as elucidating the host genetic factors that contribute to susceptibility may aid in the development of novel diagnostics and treatments for this

devastating disease. In addition, DNA sequencing costs continue to decrease dramatically, opening the door to many types of proj-ects previously impossible due to financial constraints. For exam-ple, it is now reasonable to consider projects that involve genome sequence analysis of many hundreds or more GAS strains, similar to the 1,000–human genome project that is well underway (http://www.1000genomes.org/). Finally, we are hopeful that the new ave-nues of basic and translational research made possible by molecular pathogenomics studies will ultimately provide strategies for ame-liorating human morbidity and mortality caused by GAS.

AcknowledgmentsWe thank K. Stockbauer for assistance with figures and members of our laboratories and anonymous reviewers for suggestions to improve the manuscript. The restricted length of the review pro-hibited us from citing all relevant work.

Address correspondence to: James M. Musser, Center for Molecu-lar and Translational Human Infectious Diseases Research, The Methodist Hospital Research Institute, 6565 Fannin Street, Suite B490, Houston, Texas 77030, USA. Phone: (713) 441-5890; Fax: (713) 441-3447; E-mail: [email protected].

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